Systems and methods for nucleic acid processing using degenerate nucleotides

ABSTRACT

Provided herein are compositions, systems and methods for tagging molecular events, reactions, species, etc., but without the need for complex, highly diverse libraries of tagging molecules. Provided are tagging moieties that can have a smaller number, a few, or even a single original “tagging” structure that may be transformed or transformable, in situ, into a collection of larger numbers of unique tagging or “barcode” moieties.

CROSS-REFERENCE

This application is a continuation-in-part of U.S. application Ser. No.15/355,542, filed Nov. 18, 2016, which claims priority to U.S.Provisional Application No. 62/257,438, filed Nov. 19, 2015, whichapplications are entirely incorporated herein by reference.

BACKGROUND

The field of life sciences has experienced dramatic advancement over thelast two decades. From the broad commercialization of products thatderive from recombinant deoxyribonucleic acid (DNA) technology, to thesimplification of research, development and diagnostics, enabled by theinvention and deployment of critical research tools, such as thepolymerase chain reaction (PCR), nucleic acid array technologies, robustnucleic acid sequencing technologies, and more recently, the developmentand commercialization of high throughput next generation sequencingtechnologies. All of these improvements have combined to advance thefields of biological research, medicine, diagnostics, agriculturalbiotechnology, and myriad other related fields by leaps and bounds.

Analysis of chemical reactions relies upon the ability to measure,quantify and track the consumption, production, transition andtransformation of the various reactants and products involved in thosereactions. While in some cases, the reactants and their products arethemselves, readily identifiable and measurable, in many cases theanalysis benefits from the use of tagging or labeling moieties that arecoupled to the reactants and/or products to facilitate their measurementand/or identification.

In some cases, labeling or tagging moieties include more readilyidentifiable or detectable groups, molecules or chemical moieties. Thesecan include such compositions as fluorescent chemicals, charged chemicalgroups, affinity binding groups, and in some cases encoded molecules orbarcodes that include variable amounts of information within theirstructure. Examples of particularly useful barcode molecules include,for example, nucleic acid barcodes or tags that can be read out usingany of a variety of sequence identification techniques, e.g., nucleicacid sequencing, probe hybridization based assays, and the like.

Barcoding strategies have been applied to a number of tagging andidentification strategies. For example, in some cases, step wisebuilding of oligonucleotides on solid supports, e.g., beads, has beenused as an indicator of specific chemical synthesis operations in thecreation of libraries of molecules on those solid supports, e.g., in astochastic/combinatorial synthesis process, where the building blocks ofthe oligonucleotide each reflect a specific chemical synthesis operationto which a given solid support has been exposed (See, e.g., U.S. Pat.No. 5,708,153). By reading out the sequence of added nucleotides on agiven solid support, one can identify the synthetic operations and theirorder, to identify the compound synthesized on that particular solidsupport.

In still other cases, barcode oligonucleotides have been used insequencing processes to append pre-synthesized oligonucleotides of knownsequence to sequencing libraries created from different samples, suchthat each different sample has a unique barcode oligonucleotide that isattached to and read out with the sequence of the nucleic acids fromthat sample. This may allow the pooled analysis of multiple samples,where the resulting sequence information from the pool can be laterattributed back to its starting sample.

In another sequencing application, oligonucleotide barcodes have beenused in ultra high throughput partitioning systems, to co-partition longfragments of sample nucleic acids along with barcode carrying particles,where the barcodes on an individual particle are identical, but wherelibraries of particles represent a diverse barcode library. The barcodesare then coupled to sub-segments of the long starting fragments, suchthat within a given partition, all of the sub-segments of each longfragment bear the same barcode sequence. When the sub-segments aresequenced using, e.g., short-read sequencing systems, one can attributesub-segments that have the same barcode sequence to the same startinglong molecule. This allows retention of long-range sequence context ofshort sequence reads by virtue of the included barcode sequence (See,e.g., Published U.S. Patent Application Publication No. 2014-0378345,the full disclosure of which is incorporated herein by reference in itsentirety for all purposes).

In still other cases, large numbers of diverse barcodes may beintroduced into contact with collections of sample molecules, such thatthe molecules within the collection are each coupled to a differentbarcode molecule, allowing attribution of a sequence to a specificstarting molecule, regardless of how that molecule is amplified,replicated etc., before it is identified. Where samples include multiplecopies of the same type of molecule, e.g., the same nucleic acid,sequencing of the underlying molecules as well as the different barcodeattached to each molecule may allow one to count how many individualmolecules were present at the time of tagging, allowing counting ofthose starting molecules, e.g., for messenger ribonucleic acid (mRNA)expression analysis, or the like.

SUMMARY

Recognized herein are limitations associated with barcoding strategiescurrently available. For example, for all of the barcoding strategiesdescribed above, an underlying premise is the requirement of largenumbers of diverse oligonucleotide barcodes, allowing one to distinguishbetween large numbers of different results, e.g., samples, partitions,molecules, etc. Preparing, manufacturing and allocating these diverselibraries of molecules across large numbers of samples can prove to bechallenging in a number of cases.

The present disclosure provides a dramatic improvement to this approachthat can also impart efficiency, cost and other savings to the overallprocess. The devices, methods and systems of the present inventionprovide solutions to these and other challenges of the life sciences andother fields.

Provided herein are compositions, systems and methods for taggingmolecular events, reactions, species, etc., but without the need forcomplex, highly diverse libraries of tagging molecules. In particular,provided are tagging moieties that can have a smaller number, a few, oreven a single original “tagging” structure that may be transformed ortransformable, in situ, into a collection of larger numbers of uniquetagging or “barcode” moieties.

In an aspect, the present disclosure provides a method of differentiallytagging individual members of a plurality of molecular species,comprising attaching a first tagging moiety to each of a plurality ofdiscrete molecular species, the first tagging moiety comprising atransformable tagging component; and transforming the transformabletagging component attached to each of the plurality of discretemolecules to a transformed tagging component, to distinctly tag aplurality of different members of the plurality of molecular specieswith different transformed tagging components.

In some embodiments, the plurality of discrete molecular speciescomprises a plurality of discrete nucleic acid sequences; the taggingmoiety comprises an oligonucleotide segment; and the tagging componentcomprises a transformable oligonucleotide sequence.

In some embodiments, the transformable oligonucleotide sequencecomprises one or more transformable nucleotides. In some embodiments,the one or more transformable nucleotides comprise degeneratenucleotides. In some embodiments, the one or more of the one or moretransformable nucleotides comprises 2-way degeneracy. In someembodiments, the one or more of the one or more transformablenucleotides comprises 3-way degeneracy. In some embodiments, the one ormore of the one or more transformable nucleotides comprises 4-waydegeneracy.

In some embodiments, the one or more transformable nucleotides areselected from the group of inosine, deoxyinosine, deoxyxanthine,2′-deoxynebularine, 2′-deoxyguanosine, 5-nitroindole, 3-nitroindole,N6-methoxy-2,6-diaminopurine,6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one, and the non-deoxy (orribo) versions of each of the foregoing. In some embodiments, thetransformable oligonucleotide sequence comprises from 1 to 20transformable nucleotides.

In some embodiments, the tagging moiety further comprises one or moreadditional oligonucleotide segments. In some embodiments, the one ormore additional oligonucleotide segments are selected from primersequence segments, hybridization sequence segments, ligation sequencesegments, sequencer surface attachment segments, and barcode sequencesegments. In some embodiments, the one or more additionaloligonucleotide sequences comprises a primer sequence selected from arandom primer sequence and a sequencing primer. In some embodiments, theone or more additional oligonucleotide sequences comprises ahybridization sequence. In some embodiments, the hybridization sequencecomprises a poly-T sequence.

In some embodiments, the method further comprises partitioning thetagging moieties with a sample comprising nucleic acids to be analyzedprior to the attaching, and wherein the attaching comprises attachingthe tagging moieties to the nucleic acids to be analyzed.

In some embodiments, the tagging moieties comprise a poly-T sequencesegment, and the nucleic acids to be analyzed comprise mRNA molecules.

In some embodiments, the partitioning comprises partitioning anindividual cell with the tagging moieties into a partition, and whereinthe nucleic acids to be analyzed are contained within the individualcell and wherein prior to the attaching, the individual cell is lysed torelease the nucleic acids to be analyzed into the partition.

In some embodiments, the transformable oligonucleotide sequence segmentcomprises a target sequence for a sequence substitution system. In someembodiments, the sequence substitution system comprises a CRISPR enzymesystem, and the target sequence comprises a target sequence for atargeting oligonucleotide.

In some embodiments, the transforming of the method is random orsemi-random.

In another aspect, the present disclosure provides a method of analyzingnucleic acid molecules, comprising attaching an oligonucleotide segmentto a target oligonucleotide molecule to generate a taggedoligonucleotide, wherein the oligonucleotide comprises a region thatcomprises a plurality of variable complement nucleotides; replicatingthe tagged oligonucleotide to generate a replicated taggedoligonucleotide, whereby replication generates a random or partiallyrandom replicate of the region; and analyzing the replicated taggedoligonucleotide, including the random or partially random replicate, toidentify the target oligonucleotide molecule.

In some embodiments, the region comprises from 2 to 20 variablecomplement nucleotides. In some embodiments, the region comprises two ormore contiguous variable complement nucleotides.

In some embodiments, the two or more of the variable complementnucleotides are separated from each other by one or more non-variablecomplement nucleotides.

In some embodiments, the region comprises from 4 to 10 variablecomplement nucleotides.

In some embodiments, the first oligonucleotide comprises an additionalregion that comprises a plurality of variable complement nucleotides.

In another aspect, the present disclosure provides an oligonucleotidecomposition, comprising an oligonucleotide that comprises a first regionand a second region, wherein the second region comprises a fixedsequence comprising a plurality of variable complement nucleotides,which plurality of variable complement nucleotides is transformable toyield a distinct molecular tag.

In some embodiments, the first region comprises an attachment sequencefor attachment of the oligonucleotide to a nucleic acid molecule to beanalyzed. In some embodiments, wherein the attachment sequence comprisesa primer sequence. In some embodiments, the attachment sequencecomprises a poly-T sequence.

In some embodiments, the first region comprises a barcode sequence. Insome embodiments, the first region comprises a surface attachmentsequence.

In some embodiments, the second region comprises a plurality of variablecomplement nucleotides and one or more non-variable complementnucleotides.

In another aspect, the present disclosure presents a method ofquantifying nucleic acid molecules in a population of identical nucleicacid molecules, comprising mutating the population of identical nucleicacid molecules at an expected mutagenesis rate to create a population ofdifferent mutated nucleic acids; sequencing the distinct mutated nucleicacid molecules; and computing a quantification of the nucleic acidmolecules in the population of identical nucleic acid molecules basedupon a number of different mutated nucleic acid molecules.

In some embodiments, the computing comprises quantifying the nucleicacid molecules in the population of identical nucleic acid moleculesbased upon the number of different mutated nucleic acid molecules andthe mutagenesis rate.

In some embodiments, the sequencing comprises generate sequencing readsfrom the distinct mutated nucleic acid molecules.

In some embodiments, the computing comprises computing a comparison ofthe sequencing reads to quantify the nucleic acid molecules in thepopulation of identical nucleic acid molecules.

In another aspect, the present disclosure presents a method ofdifferentiating amplification products from two or more identicalnucleic acid molecules, comprising subjecting the two or more nucleicacid molecules to mutagenesis to produce two or more mutated nucleicacid molecules; amplifying the two or more mutated nucleic acidmolecules to generate amplified mutated nucleic acid products; andsequencing the amplified mutated nucleic acid products.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 provides a schematic illustration of a tagging construct and itsimplementation, in accordance with the present disclosure;

FIG. 2 provides a high level flow chart of an example tagging process ofthe present disclosure;

FIG. 3 provides a schematic illustration of a partitioning system andprocess for allocating tagging moieties to individual cells in a taggingprocess of the present disclosure; and

FIG. 4 provides a schematic illustration of a tagging process of thepresent disclosure for use in, e.g., the quantification of messengerribonucleic acid (mRNA) expressed from genes within cells.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The term “sample,” as used herein, generally refers to a biologicaltissue, cells or fluid. Such sample may include, but is not limited to,sputum, blood (e.g., whole blood), serum, plasma, blood cells (e.g.,white cells), tissue, nipple aspirate, core or fine needle biopsysamples, cell-containing body fluids, free floating nucleic acids,urine, peritoneal fluid, and pleural fluid, or cells there from. Asample may be a cell-free (or cell free) sample. A sample may includeone or more cells.

The term “nucleic acid,” as used herein, generally refers to a monomericor polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs or variants thereof.A nucleic acid molecule may include one or more unmodified or modifiednucleotides. Nucleic acid may have any three dimensional structure, andmay perform any function, known or unknown. The following arenon-limiting examples of nucleic acids: ribonucleic acid (RNA),deoxyribonucleic acid (DNA), coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer ribonucleic acid (RNA),ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA),micro-RNA (miRNA), ribozymes, complementary deoxyribonucleic acid(cDNA), recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. Nucleic acid may comprise one or moremodified nucleotides, such as methylated nucleotides and nucleotideanalogs, such as peptide nucleic acid (PNA), Morpholino and lockednucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid(TNA), 2′-fluoro, 2′-OMe, and phosphorothiolated DNA. A nucleic acid mayinclude one or more subunits selected from adenosine (A), cytosine (C),guanine (G), thymine (T) and uracil (U), or variants thereof. In someexamples, a nucleic acid is DNA or RNA, or derivatives thereof. Anucleic acid may be single-stranded or double stranded. A nucleic acidmay be circular.

The term “nucleotide,” as used herein, generally refers to a nucleicacid subunit, which may include A, C, G, T or U, or variants or analogsthereof. A nucleotide can include any subunit that can be incorporatedinto a growing nucleic acid strand. Such subunit can be an A, C, G, T,or U, or any other subunit that is specific to one or more complementaryA, C, G, T or U, or complementary to a purine (i.e., A or G, or variantor analogs thereof) or a pyrimidine (i.e., C, T or U, or variant oranalogs thereof). A subunit can enable individual nucleic acid bases orgroups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, oruracil-counterparts thereof) to be resolved.

General

Transformable tagging groups as described herein may be employed in avariety of useful contexts. For example, they may be used to impart alevel of tag diversity, in situ, without requiring that level ofdiversity in the originating tag reagent. Additionally, they may beemployed as indicators of replication cycles, as random differentiationtags, as part of a process for creating highly diverse barcodelibraries, as unique molecular identifier molecules in certain types ofanalyses, such as molecular counting applications (e.g., for expressionanalysis, to increase confidence in variant calls in nucleic acids;e.g., by counting molecules supporting a given allele, and by takingconsensus amongst short reads with a common molecular identifier toimprove sequencing accuracy, as well as determination of copy numbervariations), as tracking tags for tracking lineages in populations,e.g., for phylogenetic reconstruction, as indicators of enzyme activity,or indicators of proximity or interaction between multiple molecules. Avariety of other uses will be apparent to those of skill in the art uponreading this disclosure.

In an application, these transformable tagging moieties may be employedas individual molecule tags for use in molecular quantitation processes.In many applications, unique molecular identifier tags have been used totag individual molecules in order to be able to individually identifyseparate starting molecules in order to quantify them. In an example, itcan be desirable to be able to quantify the number of separate messengerribonucleic acid (mRNA) molecules from a given gene in a cell or othersample, in order to be able to measure the expression levels of thatgene, either generally, or in response to some stimulus, e.g., a drugcandidate or other environmental stimulus. In this context, discretecopies of mRNA molecules within a cell, that are expressed from a givengene, may be stochastically tagged with different nucleic acid barcodemolecules, such that each discrete molecule has a unique identifiersequence attached to it, or a unique molecular identifier (“UMI”).Because each starting mRNA molecule expressed from a given gene now hasa UMI sequence attached to it, it can be subjected to rounds ofamplification, without losing the information as to the number ofstarting molecules, e.g., each different UMI attached to mRNA denotes aseparate starting molecule. Amplification allows for greatly simplifieddetection, e.g., using nucleic acid arrays that target the genes ofinterest or the UMIs, nucleic acid sequencing, or other approaches.Following amplification, detection of the different UMIs present allowsthe inference of the number of starting mRNA molecules for a given gene,and thus an inference of expression of that gene. Examples of this typeof use of UMIs are described in, e.g., “Counting Absolute Numbers ofMolecules Using Unique Molecular Identifiers”, Kivioja, et al., NatureMethods 9, 72-74 (2012), the full disclosure of which is incorporatedherein by reference in its entirety for all purposes.

While useful in some contexts, it will be appreciated that these methodsmay be reserved for samples with relatively small numbers of molecules,as the requisite tagging library may rapidly increase in complexity andcost, as the number of molecules in a sample increase. Restated, as thenumber of molecules to be counted increases, it may result in anecessary and substantial increase in the number of different taggingmoieties that may be required to be applied to the sample, in order toget unique molecular tagging. Likewise, as the number of different genesto be analyzed increases, it increases the required complexity of theUMI library. Moreover, biochemistries for the creation, ligation orother attachment, replication, etc. of these diverse libraries cannot beoptimized for any particular sequence, but may be optimized for theaverage sequence, which will typically result in optimization for noneof the actual sequences used.

As described herein, however, a relatively simple and constant taggingstructure may be used that incorporates transformable moieties, asdescribed above, within the tagging moiety in order to impart diversity,in situ, to the tagged molecules. In particular, one may employ atagging moiety that has a single, but transformable tagging moiety,where subsequent processing of tagged molecules will transform thetagging molecule in a random or semi-random way, to impart diversity tothe tagging groups in a sample, where that level of diversity did notexist originally. This allows one to use a small number, a few or even asingle transformable tagging moiety in an analysis in place of muchlarger numbers of unique barcode molecules required by prior processes,as the diversity required for a given analysis will be introduced uponrandom or semi-random transformation of the tag.

In the context of the expression analysis example, above, in place ofthe diverse library of nucleic acid UMIs that are individually andstochastically attached to separate mRNA molecules, one may attach asingle, few, or relatively small number of transformable taggingmoieties to the different mRNAs. Following a single round ofreplication, each copy of mRNA for a given gene may be replicated with arandom or semi-random sequence tag attached, that by virtue of therandomness or semi-randomness of the replication process for the tag,may yield a differently tagged replicate for each starting molecule. Bythen detecting and counting the number of different transformed taggingmoieties, one may infer the number of starting mRNA molecules.

Transformable Tagging Moieties

The present disclosure provides “tagging” moieties that includetransformable elements that may be converted into a desired taggingstructure after they are associated with the component, which they areintended to tag. In some cases, these transformable structures maypossess a common structure, but are transformed into a diverse set ofstructures after the transformation process, e.g., where a population oftags having a single structure is transformed into a diverse populationof different structures. In some cases, these transformable groups aretransformed into random or semi-random resultant moieties to impartdiversity to the tagged molecules which can be identified and used inthe characterization of, e.g., a reaction, its reactants and/or itsproducts.

While specific examples of transformable tagging moieties are describedin terms on nucleic acids, polynucleotides, etc., it will be appreciatedthat other transformable tagging moieties may be employed. For example,transformable moieties may comprise nucleic acids (e.g., nucleotides,oligonucleotides, polynucleotides, including ribonucleotides anddeoxyribonucleotides, as well as analogs of these, such as dideoxyribonucleotides, degenerate nucleotides, etc.), polypeptides (e.g.,proteins, enzymes, polypeptides, oligopeptides, etc.), carbohydrates(e.g., dextrans, starches, celluloses, etc.), organic compounds,fluorophores, chromophores, colloidal elements, particles, beads, or thelike, where a first structure may be transformed into one or more secondstructures upon implementation of a process operation, in order to gaindiversity of tagging moieties in a reaction.

In an example, a transformable moiety may include a transformableoligonucleotide sequence, where during replication, translation,transcription, or other transformation processes, the nucleotides ofsuch sequence (also referred to herein as “bases” for simplicity) in thesequence are transformable, in situ, to varied or variable resultingspecies. A variety of different mechanisms may be used to transformnucleotides in a sequence, in situ, including, for example, the use ofdegenerate bases, e.g., bases for which complementary base pairing mayvary, sequence segment based transformation, e.g., removing andreplacing sequence segments, as well as chemical transformations ofindividual bases or sequences of bases, e.g., oxidative deamination ofbases, or other chemical modifications (e.g., treatment with nitrousacid or alkylating agents), exposureto ionizing radiation, treatmentwith enzymes that modify bases (e.g., adenosine deaminase, cytosinedeaminase, xanthine oxidase, editosomes), that change base pairing orprocesses that cause template driven or non-template drive insertion oraddition, such as M-MLV reverse transcriptases, terminaldeoxynucleotidyl transferases, or transposons that catalyze their owninsertion.

In certain cases, the transformable nucleotides may include nucleotidesthat are subject to random or semi-random “complement” incorporation,which nucleotides, or bases, may also be referred to herein as variablecomplement nucleotides or bases. In particular, during oligonucleotidereplication, transcription or translation, faithful processing by theinvolved enzymes or enzyme systems typically incorporates a single typeof complementary building block in response to encountering a givennucleotide or set of nucleotides. For example, template driven,polymerase mediated nucleic acid replication using typical faithful DNApolymerase enzymes, e.g., a DNA polymerase replicating a given DNAstrand, when it encounters one type of nucleotide, will typicallyincorporate a single specific type of complementary nucleotide. Forexample, when encountering a purine adenosyl (A) or guanidyl (G)nucleotide in the sequence, a polymerase will typically incorporate apyrimidine thymidyl (T) or cytosyl (C) nucleotide as the complementarybase in the sequence, respectively, and vice versa. Thus, a typicalbarcode sequence made up of these bases may typically be replicated intothe same complementary structure substantially every time by thefaithful polymerase enzyme. In accordance with some aspects of thepresent disclosure, however, a barcode segment may include one or morenucleotides that are capable of having random or semi-randomcomplements, such that when replicated, they produce random orsemi-random replicate sequences in response. As will be appreciated,random incorporation may likewise be driven through the use of a lowerfidelity polymerase enzymes toward conventional bases, ornon-proofreading enzymes, e.g., having substitution rates of greaterthan 0.1%, and in some cases greater than 1%, greater than 5% or evenhigher. Examples of such low fidelity polymerases include, e.g., FamilyY polymerases, translesion synthesis polymerases, Escherichiacol/polymerases IV and V, human polymerases; ζ, π, τ, κ and Rev1, aswell as modified versions of polymerases having reduced or noproofreading capability, such as phi29 mutant enzymes, phi29 N62D andother non-proofreading mutants (e.g., as described in Korlach et al.,Methods in Enzymology, Real-Time DNA Sequencing from Single PolymeraseMolecules, (2010) 472:431-455), low-fidelity mutants of pfu-Pol (see,e.g., Biles et al., Nucl. Acids Res. 32(22):e176 2004), viralpolymerases such as DNA Polymerase X (Pol X) from African Swine FeverVirus (ASFV), Such polymerases may be used alone or in combination withtransformable bases as described elsewhere herein, or may be used inconjunction with particular sequence motifs for which these polymerasesdemonstrate higher base substitution rates. In some cases, a single typeof polymerase may be used in achieving the transformation of the taggingsequence. Conversely, in other cases, mixes of different polymeraseenzymes having different responses to different degenerate bases, may becombined in single reaction mixture to increase diversity or otherwisebetter control the transformation process.

A variety of nucleotide or nucleotide like moieties have been describedthat have random or semi-random complements when replicated inpolymerase reactions, e.g., during replication, they may be complementedwith two or more different nucleotides in the produced or “replicate”strand. For ease of discussion, these bases are referred to herein asdegenerate bases. For example, a number of bases are able to interactwith a polymerase in a way which will be unbiased enough to at leastprovide two-way degeneracy in replicating at that base, i.e., able toincorporate two or more different nucleotides in response to and as a“complement” to such bases. In some cases, bases that result inpolymerase incorporation at a level of at least 2-way, at least 3-way,or even 4-way degeneracy may be used. Generally, as used herein,degeneracy generally refers to bases that under particular reactionconditions, e.g., using a particular polymerase with particularnucleotide, buffer, and salt concentrations, etc., will exhibit unbiasedincorporation, e.g., will incorporate a different nucleotide in responseto a degenerate base, in at least 1% of the instances in which itencounters such degenerate base, at least 5% of the instances, in somecases, at least 10% of the time, in some cases a least 20% of the time,in some cases at least 30% of the time, in some cases at least 40% ofthe time, and in some cases, at least 50% of the time. For example, andsolely for ease of discussion, a transformable nucleotide may exhibittwo way degeneracy if it results in different base incorporations, e.g.,at least 5% of the time, e.g., if it incorporates an A 5% of the time,and a G the other 95% of the time.

As alluded to above, in some cases, concentrations of variousnucleotides within the polymerization reaction mixture may be adjustedto provide a desired degeneracy rate in a given reaction. For example,to even out incorporation of different bases, one may adjust theirrelative concentrations to increase incorporation rate of one whiledecreasing the relative incorporation rate of another in response to agiven transformable or degenerate base. As such, one may provide eventwo way, three way or four way degeneracy at a given degenerate base byproviding the various nucleotide reagents at concentrations that yieldan equivalent incorporation rate of each at the particular degeneratebase.

As will be appreciated, bases that result in “complement” incorporationthat exhibits the above-described degeneracy will be characterized asbeing random or semi-random. For example, in some cases, a defined biastoward a subset of complement bases, e.g., only purines, or onlypyrimidines, may be identified as being semi-random, where completeindiscriminate complement paring of a given base may be viewed as beingcompletely random.

Examples of such transformable nucleotides may include, e.g., inosine,deoxyinosine, deoxyxanthine, 2′-deoxynebularine, 2′-deoxyguanosine,5-nitroindole, 3-nitroindole, N6-methoxy-2,6-diaminopurine,6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one, and the non-deoxy (orribo) versions of each of the foregoing. For these bases, one mayprovide for a variety of incorporation patterns using one or more ofthese bases as transformable bases within a transformable taggingsequence, depending upon the transformable bases used. For example, sometransformable bases, such as inosine, while displaying levels ofdegeneracy, may nonetheless display a stronger preference to complementwith, and therefor drive the incorporation of one type of nucleotide,e.g., cytosine nucleotide (C). Other transformable bases, like5-nitroindole, may show more balanced 4-way degeneracy, e.g., an abilityto incorporate any of the four natural bases, e.g., AGCT, in response.In another example, deoxyxanthine, while displaying 4-way degeneracy, insome cases displays a stronger preference for complementing withpyrimidine nucleotides, e.g., T or C.

A tagging or barcode sequence including one or more of these degeneratebases may be employed by appending the tagging sequence segment to atarget nucleic acid or nucleic acid fragment of interest. Uponpolymerase mediated replication of the target nucleic acid, the taggroup will also be “replicated”, but that replication may incorporaterandom or semi-random complement bases to the degenerate base positionsto create a unique or semi-unique tag appended to the replicatemolecule. As a result, a single sequence of degenerate bases can giverise to a number, and potentially a large number of different tagsequences upon polymerase replication.

As will be appreciated, the transformable oligonucleotide taggingsequences may include degenerate bases in addition to non-degeneratebases, or they may include all degenerate bases. Likewise, thedegenerate bases included may have two way degeneracy, three-waydegeneracy or four-way degeneracy, and/or may have certain preferencesdespite their level of degeneracy. In some cases, degenerate bases maybe interspersed with non-degenerate bases, or two-way degenerate basesmay be interspersed with three and/or 4 way degenerate bases in randomor even known or predetermined patterns. Use of known or predeterminedpatterns may permit the ready identification of the tag sequences byvirtue of their reflection of a known or predetermined patternreflective of the pattern of degenerate bases and/or non-degeneratebases included.

In some cases, and depending upon a desired level of possible diversity,the number of degenerate bases in a given tagging sequence may vary from1 to 100 or more, from 1 to 20 transformable bases, from 1 to 10transformable bases, from 1 to 5 transformable bases, or anyintermediate number of transformable bases within any of the foregoingranges.

Moreover, these transformable bases may, as noted previously, becontiguous within a sequence segment, or they may be interspersed withnon-degenerate bases. Such interspersed bases may separate individualtransformable bases from other transformable bases within the taggingsequence, or they may separate pairs, groups or subsets of transformablebases from other individual, pairs, groups or subsets of transformablebases. These interspersed transformable bases may, likewise, be presentis individual bases in the sequence, or as contiguous pairs, groups orsubsets of non-transformable bases in the tagging sequence.

As will be appreciated, one may select the level of potential diversityfor a transformable tagging sequence through selection of the number ofdegenerate or transformable bases in a tagging sequence, and the levelof degeneracy for each such base. Moreover, as discussed above, one canintroduce a level of additional diversity by providing sets oftransformable tagging segments with varying sequences of transformablenucleotides, e.g., by shuffling the order to the degenerate bases usedin a library of tagging molecules. Such selection can be motivated byany of a number of requirements or desires, including, the level ofdiversity required or desired for any given application, e.g., thenumber of expectant molecules to be tagged in a molecular countingapplication, as well as the desire to be able to identify taggingsequences from a higher level signature, e.g., resulting from their semirandomness. For example, one may select the transformable bases in atagging sequence to reflect a general pattern of resulting sequences,e.g., localizing purine or pyrimidine specific transformable bases atcertain positions, as well as non-degenerate bases interspersed amongother transformable bases. By incorporating patterns of semi-randomtransformable bases or overall sequences, one may be able to betteridentify sequences that more likely result from the tagging sequence.

In other cases, the transformable tagging moiety may include a segmentthat is transformed in whole, as opposed to on a building block bybuilding block basis. For example, in some cases, an original taggingmoiety may be provided that presents a target for insertion of areplacement sequence segment that yields a desired level of diversitywhile starting from a common original tagging segment. An example ofsuch an approach may include the use of a targeted mutagenesis mechanismwhere a transformable sequence segment may be transformed (e.g., alteredor replaced, in whole or in part). For example, a tagging sequencesegment may form the basis of a target sequence for a targeted sequencereplacement system. For example, a transformable tagging sequencesegment may be targeted using, e.g., a guide RNA associated with aCRISPR associated RNA guided DNA endonuclease enzyme, such as Cas9, thatis able to target a specific sequence through the guide RNA, and excisethat sequence (See, e.g., Genome Engineering Using the CRISPR-Cas9System, Ran, et al., Nature Protocol, (2013), 8(11):2281-2308). Onceexcised, replacement sequence segments may be readily inserted by using,e.g., complementary flanking regions that allow ligation of the newsequence segment at the point of excision of the prior transformablesequence segment, using, e.g., conventional ligation biochemistries oremploying, e.g., non-homologous end joining (NHEJ) or homology-directedrepair (HDR). As will be appreciated, a variety of other targetedediting nucleases may be used in a similar fashion, e.g., including forexample, zinc finger nucleases (ZFNs), and transcription activator-likeeffector nucleases (TALENs), see, e.g., Porteus MH, Baltimore D.Chimeric nucleases stimulate gene targeting in human cells. Science.2003; 300:763; Miller J C, et al. An improved zinc-finger nucleasearchitecture for highly specific genome editing. Nat. Biotechnol. 2007;25:778-785; Sander J D, et al. Selection-free zinc-finger-nucleaseengineering by context-dependent assembly (CoDA). Nat. Methods. 2011;8:67-69; Wood A J, et al. Targeted genome editing across species usingZFNs and TALENs. Science. 2011; 333:307; Christian M, et al. TargetingDNA double-strand breaks with TAL effector nucleases. Genetics. 2010;186:757-761; Zhang F, et al. Efficient construction of sequence-specificTAL effectors for modulating mammalian transcription. Nat. Biotechnol.2011; 29:149-153; Hockemeyer D, et al. Genetic engineering of humanpluripotent cells using TALE nucleases. Nat. Biotechnol. 2011;29:731-734.

In some cases, a transformable tagging moiety, e.g., a transformablesequence may comprise a sequence that is more susceptible and/or subjectto chemical or UV mutagenesis in order to drive transformation of thetagging segment or even transformation of the sequence segment ofinterest, such that such mutagenesis results in sufficient diversity fora given analysis. For example, if counting identical sequences, one maymutagenize such sequences in a manner that is expected to impact eachand every molecule. Subsequent analysis of those sequences, may allowone to determine the number of staring molecules based upon the numberof differently mutated sequences. Such mutagenesis may in some cases,again, be targeted using, e.g., targeting or guide oligonucleotideprobes, or it may be random, e.g., non-targeted.

Structures

Also provided herein are compositions that include oligonucleotides thatcomprise as a part of their sequences, the tagging oligonucleotidesequences or segments described elsewhere herein. These compositions mayinclude these oligonucleotides alone, or in conjunction with othercomponents, including without limitation, buffers, salts, reactants,enzymes, sample components, e.g., cells, tissues or other sampleconstituents, solid supports, such as particles, beads, hydrogel beads,array surfaces, etc.

The tagging moieties described herein may include additional elementswithin their larger structure, e.g., to impart additional functionalityto the tagging moieties. For example, such structures may includeadditional elements that may provide functions within an application ofthe tagging moiety or for the resulting tagged reactant.

By way of example, the tagging moieties described may be provided withinstructures that facilitate their attachment or appending to otherreactants. For example, they may include activatable chemical groupsthat can facilitate chemical coupling with other groups, affinitybinding portions, e.g., avidin, streptavidin, biotin, etc., for affinityattachment, or they may include other mechanisms allowing for thiscoupling.

By way of example, oligonucleotide tagging moieties, as described above,may include additional sequence segments that permit their attachment orappending to other sequence segments, e.g., target sequence segments. Aswill be appreciated, attachment or appending of a tagging moiety, e.g.,a tagging oligonucleotide, to another species, e.g., a target nucleicacid sequence or portion thereof, includes a variety of differentattachment or appending approaches. For example, in some cases,attachment of a tagging oligonucleotide to another sequence segment maycomprise covalent attachment via, e.g., ligation attachment to a 3′ or5′ end of the other sequence segment, or through covalent cross-linkingor other side chain attachment to the other sequence segment.

Additionally, attachment may include non-covalent attachment to theother sequence segment, e.g., through affinity coupling, such as throughhybridization of a portion of the tagging oligonucleotide to thetargeted sequence segment, or through other affinity mechanisms forother molecular species, e.g., through antibody/antigen coupling, avidinor streptavidin/biotin coupling, or through association with specificassociation groups, e.g., association peptides, and the like.

In still other aspects, attachment of a tagging oligonucleotide may bethrough the priming and extension of primer sequences that are includedwithin the tagging oligonucleotide structure, such that a complement ofthe targeted sequence segment is attached to the extended primer/taggingoligonucleotide. As will be appreciated, the tagging oligonucleotide andthe sequence segment, when referred to as attached, will interchangeablyrefer to the complements or replicates of either or both sequencesegments. Accordingly, and as will be appreciated, attachment willinclude both the attachment of a tagging moiety to a sequence segment,as well as attachment of the tagging oligonucleotide to a complement ofthe sequence segment, as well as attachment of a complement to theoriginal tagging oligonucleotide to a sequence segment, its complementor a further complement of such complement (i.e., a replicate of anoriginal sequence segment).

The additional sequence segments may comprise hybridization probes forattaching to the target sequences by hybridization, or they may includeprimer sequences that are capable of annealing to the target sequencesegments, such that extension of the primer segment replicates acomplement to the target sequence into the extension product thatincludes the tagging sequence, which for purposes of the presentdisclosure constitutes attachment or appending of the tagging moleculeto the target, as used herein.

In some cases, the tagging moieties may include sequence overhangsand/or bridging or splint sequences in order to facilitate ligation orother coupling of the tagging moiety to a given sequence.

Priming or hybridization sequences may be constructed to anneal tospecific sequences within a target sequence, or they may be constructedto anneal to random portions of target sequences, e.g., as universalprimers, such as random n-mer sequences, such that different primersequences on different tagging oligonucleotides may prime at differentlocations within a target sequence.

An example of a tagging oligonucleotide and its use in tagging a segmentof a target sequence is illustrated in FIG. 1. As shown, anoligonucleotide 100 includes a tagging segment 102 that comprises one ormore degenerate bases (Z) within its sequence. The one or moredegenerate bases may be in a given region of the oligonucleotide 100. Insome cases, the oligonucleotide 100 includes at least 2, 3, 4, 5, 6, 7,8, 9, or 10 regions each with one or more degenerate bases. As notedabove, although illustrated as including a number of contiguousdegenerate bases, the tagging sequence may, in some cases, include oneor more non-degenerate bases within its sequence. Likewise, althoughshown as a 10-mer tagging sequence, or a tagging sequence including 10degenerate bases, the tagging sequence may be longer or shorter, andinclude more or fewer degenerate bases, as described elsewhere herein.

The tagging sequence may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90 or 100 degenerate bases. The taggingsequence may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 nucleotides.

Oligonucleotide 100 is also shown as including a priming sequence 104 atits 3′ terminus, for annealing to and priming replication of a targetsample nucleic acid fragment 106. As will be appreciated, this primingsequence may be specific to a sequence within the target sequence ofinterest, it may be a random priming sequence, e.g., an n-mer, or it maybe targeted to a particular type of sequence segment, e.g., to anneal toa poly-adenylated terminus (poly-A tail) of a mRNA molecule, or othercommon sequence type. Also as shown, oligonucleotide 100 may includeadditional nucleic acid segments, such as additional barcode segment108, sequencing primer segment 110, as well as sequencer specificattachment sequences (not shown). For example, the oligonucleotide 100may include a flow cell sequence for use in massively parallelsequencing (e.g., Illumina sequencing).

As shown, tagging oligonucleotide 100 anneals to a target sequence ofinterest and is used to prime extension and complementary replication ofthat target sequence, resulting in the tagging oligonucleotide 100 beingappended to the complement replicate segment 120 of the target sequence106. Upon subsequent replication of tagged segment 102 in taggingoligonucleotide 100, the transformable nature of tagging segment 102replicates into a random or semi-random sequence segment 122 attached toa copy 106′ of the original target sequence segment 106. The resultantrandom or semi-random segment 122 may be different for differentmolecules in the sample, despite originating from the same taggingoligonucleotide sequence segment 102 in tagging oligonucleotide 100.

As noted above, a number of other structures may be included along withthe transformable tagging sequence segments described above. Forexample, in some cases, the transformable tagging segments may beincluded in an oligonucleotide structure along with other tagging orbarcoding structures. Examples of particularly useful barcodingoligonucleotides are described in, e.g., Published U.S. PatentApplication Publication Nos. 2014/0378345, 2014/0228255, 2015/0376700,2015/0376605, and 2016/0122817, the full disclosures of which are herebyincorporated herein by reference.

The barcodes can have a variety of structures. In some cases, barcodesare a part of an adapter. Generally, an “adapter” is a structure used toenable attachment of a barcode to a target polynucleotide. An adaptermay comprise, for example, a barcode, polynucleotide sequence compatiblefor ligation with a target polynucleotide, and functional sequences suchas primer binding sites and immobilization regions. In some cases, anadapter is a forked adapter.

In some cases, these barcodes may be used to tag sequence segmentfragments that have been co-partitioned into, e.g., submicroliterdroplets (nanoliter or picoliter scale droplets). Such sequence segmentsmay be derived from solutions of sample nucleic acids or from individualcells that are co-partitioned with the barcodes for tagging.Additionally or alternatively, the additional tagging or barcodingstructures may include a separate barcode reflective of the specificsample from which the nucleic acids were derived, in order to allowsubsequent differentiation of nucleic acids from different sample on apooled sequencing run.

In some cases, the tagging oligonucleotides described herein, includingany additional sequence segments, may be provided as elements of alarger oligonucleotide library. For example, the tagging oligonucleotidesegments are incorporated into barcode oligonucleotide libraries, suchas those described in Published U.S. Patent Application Publication No.2014/0228255, incorporated herein by reference in its entirety for allpurposes.

Random methods of polynucleotide synthesis, including random methods ofDNA synthesis can be used to generate barcode oligonucleotide libraries.During random DNA synthesis, any combination of A, C, G, and/or T may beadded to a coupling operation so that each type of base in the couplingoperation is coupled to a subset of the product. If A, C, G, and T arepresent at equivalent concentrations, approximately one-quarter of theproduct will incorporate each base. Successive coupling steps, and therandom nature of the coupling reaction, enable the generation of 4^(n)possible sequences, where n is the number of bases in thepolynucleotide. For example, a library of random polynucleotides oflength 6 may have a diversity of 4⁶=4,096 members, while a library oflength 10 may have diversity of 1,048,576 members. Therefore, very largeand complex libraries can be generated. These random sequences may serveas barcodes. Any suitable synthetic bases may also be used. In somecases, the bases included in each coupling operation may be altered inorder to synthesize a preferred product. For example, the number ofbases present in each coupling operation may be 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more. In some cases, the number of bases present in eachcoupling operation may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore. In some cases, the number of bases present in each couplingoperation may be less than 2, 3, 4, 5, 6, 7, 8, 9, or 10. Theconcentration of the individual bases may also be altered in order tosynthesize the preferred product. For example, any base may be presentat a concentration of about 0.1, 0.5, 1, 5, or 10-fold the concentrationof another base. In some cases, any base may be present at aconcentration of at least about 0.1, 0.5, 1, 5, or 10-fold theconcentration of another base. In some cases, any base may be present ata concentration of less than about 0.1, 0.5, 1, 5, or 10-fold theconcentration of another base. The length of the random polynucleotidesequence may be any suitable length, depending on the application. Insome cases, the length of the random polynucleotide sequence may be 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or morenucleotides. In some cases, the length of the random polynucleotidesequence may be at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or more nucleotides. In some cases, the length of therandom polynucleotide sequence may be less than 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some cases,the library is defined by the number of members. In some cases, alibrary may comprise about 256, 1024, 4096, 16384, 65536, 262144,1048576, 4194304, 16777216, 67108864, 268435456, 1073741824, 4294967296,17179869184, 68719476736, 2.74878*10¹¹, or 1.09951*10¹² members. In somecases, a library may comprise at least about 256, 1024, 4096, 16384,65536, 262144, 1048576, 4194304, 16777216, 67108864, 268435456,1073741824, 4294967296, 17179869184, 68719476736, 2.74878*10, or1.09951*10¹² members. In some cases, a library may comprise less thanabout 256, 1024, 4096, 16384, 65536, 262144, 1048576, 4194304, 16777216,67108864, 268435456, 1073741824, 4294967296, 17179869184, 68719476736,2.74878*10, or 1.09951*10¹² members. In some cases, the library is abarcode library. In some cases, a barcode library may comprise at leastabout 1000, 10000, 100000, 1000000, 2500000, 5000000, 10000000,25000000, 50000000, or 100000000 different barcode sequences.

The random barcode libraries may also comprise other polynucleotidesequences. In some cases, these other polynucleotide sequences arenon-random in nature and include, for example, primer binding sites,annealing sites for the generation of forked adapters, immobilizationsequences, and regions that enable annealing with a targetpolynucleotide sequence, and thus barcoding of the polynucleotidesequence.

In many cases, such libraries may be provided tethered to beads orparticles for use in efficient delivery of the library elements. Forexample, in some cases, beads are provided having the taggingoligonucleotide structures described above, attached to them. In suchcases, an individual bead may include oligonucleotides that include afirst region that includes a transformable oligonucleotide sequence,e.g., as a transformable sequence, or sequence of transformablenucleotides. As noted above, this first region may be common to all ofthe oligonucleotides attached to a given bead, or among populations ofbeads. Alternatively, the first region may vary among different beads ordifferent populations of beads. The oligonucleotides may also includeadditional regions or sequence segments, e.g., second, third, fourth,etc. regions, where such additional regions may include variableregions, e.g., that vary in sequence as between oligonucleotides ondifferent beads. Such variable regions may include barcode sequencesthat differ among oligonucleotides on different beads. By providing abead with a given barcode sequence segment, but where such barcodesequence differs on other beads, one can readily partition differentbarcodes into different partitions by merely partitioning the beads onan individual basis. Examples of partitioning methods for such barcodesequences are described in, e.g., Published U.S. Patent ApplicationPublication Nos. 2014/0378345, and 2015/0292988, the full disclosures ofeach of which are incorporated herein by reference in their entirety forall purposes.

Partitioning methods can include flowing an aqueous fluid comprising asuspension of barcode sequences into a droplet generation junctioncomprising a partitioning fluid. During a window of droplet generation,the barcode sequences can be flowing into the droplet generationjunction at a frequency that varies less than 30%. The method can alsoinclude partitioning the barcode sequences in the partitioning fluidduring the window of droplet generation. Another partitioning method caninclude providing a gel precursor in an aqueous fluid and flowing theaqueous fluid having the gel precursor through a fluid conduit that isfluidly connected to a droplet generation junction comprising apartitioning fluid. The partitioning fluid can comprise a gel activationagent. The method can also include forming droplets of the aqueous fluidin the partitioning fluid, where, within the droplets, the gelactivation agent contacts the gel precursor to form gel microcapsules.

Other variable regions may be provided within the oligonucleotidesequences, e.g., for use as random n-mer priming sequences, where suchvariability may exist as between oligonucleotide sequences on a givenbead, as between individual beads, or as between bead populations.Likewise, the oligonucleotide sequences may include other commonregions, such as common primer sequences, e.g., sequencer specificprimer sequences, attachment sequences, and the like, that are common asto oligonucleotides on a given bead, as between two or more beads or asamong a population of beads.

The tagging oligonucleotides may be attached to the beads through, e.g.,a reversible or cleavable linkage, such that the oligonucleotides may beseparated from the beads upon application of a stimulus, e.g., achemical, thermal, optical, or mechanical stimulus. Examples of suchcleavable linkages include, e.g., those described in Published U.S.Patent Application Publication Nos. 2014/0228255 and 2014/0378345, eachof which is entirely incorporated herein by reference. In some cases,the beads themselves may comprise degradable structures, such asdegradable polymers or hydrogels that may degrade to further facilitatethe release of the oligonucleotides from the beads into a substantiallyhomogenous reaction mixture. See, e.g., U.S. Patent ApplicationPublication Nos. 2014/0228255 and 2014/0378345 each of which is entirelyincorporated herein by reference.

Processes

Although described in some detail above for use in quantifying nucleicacid molecules, e.g., mRNA for expression analysis, the followingexample provides one detailed example of one type of specific processfor employing transformable tagging moieties in such expressionanalysis.

In an exemplary process for evaluating expression of one or more genesin cell cultures, one may individually analyze the contents of the cellsusing processes, e.g., as described in U.S. Patent ApplicationPublication No. 2015/0376609, which is incorporated herein by referencein its entirety for all purposes, and using the transformable taggingmoieties described herein.

Methods of analyzing nucleic acids from cells include providing nucleicacids derived from an individual cell into a discrete partition;generating one or more first nucleic acid sequences derived from thenucleic acids within the discrete partition, which one or more firstnucleic acid sequences have attached thereto oligonucleotides thatcomprise a common nucleic acid barcode sequence; generating acharacterization of the one or more first nucleic acid sequences or oneor more second nucleic acid sequences derived from the one or more firstnucleic acid sequences, which one or more second nucleic acid sequencescomprise the common barcode sequence; and identifying the one or morefirst nucleic acid sequences or one or more second nucleic acidsequences as being derived from the individual cell based, at least inpart, upon a presence of the common nucleic acid barcode sequence in thegenerated characterization.

For example, these processes may be used in the analysis andquantification of gene expression within individual cells, eithergenerally, or in response to certain stimuli.

In at least one approach, a set of tagging oligonucleotides may beemployed that include a common, but transformable tagging segment intheir sequence. Following tagging of individual expressed copies of oneor more genetic elements, e.g., genes or gene fragments, referred toherein as expression products, these common tagging elements may betransformed such that for each individual expressed molecule, a unique,or substantially unique tagging element is created in a tagged copy ofthe expressed molecule, to allow the unique identification of thatoriginating expressed copy. By counting the uniquely identifiableexpressed copies, one can infer the number/quantity of the expressedcopies of a given gene.

A simplified process is schematically illustrated in the flow chartprovided in FIG. 2. In particular, at stage 202, a cell may express oneor more genes in the form of messenger RNA (mRNA). At stage 204, thevarious individual mRNA molecules expressed by a given cell aresubjected to tagging with an oligonucleotide sequence that includes atransformable tagging segment. The individual tagged mRNA molecules arethen processed at stage 206 to transform the transformable taggingsegment into a new tagging segment that is unique for each startingtagged mRNA molecule, e.g., as a tagged cDNA molecule. These resultingcDNA molecules may then be subjected to amplification processes at stage208, while preserving the attribution of the resulting amplifiedmaterial to its original starting molecule (based upon the transformedtagging segment). The amplified cDNA molecules and their associated tagsmay then be sequenced at stage 210. From the sequence data, one canidentify sequences of a given gene at stage 212, and by virtue of thenumber of different tags associated with the sequences of that gene,identify or at least infer the number of starting expressed moleculesfor that gene.

In a more detailed discussion of an example process, a cell suspensionmay be subjected to co-partitioning into aqueous droplets in an oilemulsion, e.g., as described in U.S. Patent Publication No.2015-0376609, filed Jun. 26, 2015, which is entirely incorporated hereinby reference, using, e.g., a microfluidic droplet or emulsion generationsystem. A microcapsule may also be co-partitioned into the discretedroplets, where the microcapsules carry the tagging oligonucleotidescoupled to the microcapsule in a releasable fashion, e.g., allowingrelease of the tagging oligonucleotides upon application of a stimulusto the content of the droplets. As described above, the oligonucleotideswill typically comprise the transformable tagging sequence segment alongwith other functional sequence segments, e.g., other barcode segments,priming segments, attachment segments and the like. In this particularexample, the tagging oligonucleotides also comprise a barcode segmentthat will be common for all tagging oligonucleotides on a givenmicrocapsule, but which may vary among different microcapsules. Byco-partitioning a single cell with a single microcapsule, this barcodecan function as an address moiety to tag and identify all of the nucleicacids that are derived from the individual cell.

Following co-partitioning, the cells within the droplets may be lysed,e.g., through inclusion of a lysis agent within the droplet, e.g., adetergent, chaotropic agent or other lysing agent, or they may be lysedthrough the application of other stimuli, e.g., mechanical, thermal,electrical, etc. Once lysed, the contents of the cells, including themessenger RNA from expressed genes will be released into the droplets.Tagging oligonucleotides present on the co-partitioned microcapsules arealso released into the droplet and may be configured to specificallyinteract with the mRNA molecules, e.g., by using a poly-T sequencesegment as a capture/priming segment against the poly-A tail of themRNA, e.g., as segment 104 in FIG. 1.

The overall process is schematically illustrated in FIGS. 3 and 4. Asshown in FIG. 3, individual cells of interest are co-partitioned alongwith individual microcapsules bearing tagging oligonucleotides asdescribed herein, in a microfluidic channel network 300. The cellsuspension 320 is passed through a first channel segment 302 to a firstmixing junction, where it is co-mixed with a flowing suspension ofmicrocapsules or beads 322 bearing the tagging oligonucleotides, cominginto the junction 304 from another channel segment 306.

The microcapsule suspension may also include a lysis agent to be mixedwith and act upon the cells once they are partitioned. The cells andmicrocapsules are flowed at a rate that allows enough space betweenadjacent microcapsules and adjacent cells, so as to increase theprobability of co-partitioning of an individual cell with an individualmicrocapsule. The co-mixed suspension of cells and microcapsules 324 arethem driven into a droplet generation 308 junction or partitioningjunction, where they are focused by coaxial flows of oil streams comingin from side channels 310 and 312 such that individual droplets 326 ofthe aqueous co-mixed suspensions are formed in a flowing oil stream inoutlet channel segment 314.

Once co-partitioned into a droplet, an individual cell, exposed to thelysis agent, will release its contents, e.g., mRNA molecules 328, intothe droplet. Likewise, the microcapsule will release its payload oftagging oligonucleotides, e.g., tagging oligonucleotides 330, into thedroplet as well. Once present in a homogeneous mixture within a givendroplet, the tagging oligonucleotides may be used to tag fragments ofthe nucleic acids from the cells, and as described particularly here andas schematically illustrated in FIG. 4, mRNA molecules.

As shown in FIG. 4, tagging oligonucleotides 402 released from themicrocapsule, by virtue of their inclusion of a poly-T sequence 404,will anneal with the poly-A tail 406 of the mRNA 408 released from anindividual cell within the droplet. As described previously, the taggingoligonucleotides include both a common sequence of transformablenucleotides as the transformable tagging segment 410, along with abarcode segment 412 of oligonucleotides that is common to all of theoligonucleotides released from a given microcapsule. These barcodesserve to attribute resulting produced fragments as having originatedfrom the same cell when all of the nucleic acids are later sequenced.

Following annealing of the tagging oligos 402 to the mRNA molecules 408,a reverse transcriptase enzyme, present within the aqueous droplet (andintroduced with one of the cell suspension and/or the microcapsulesuspension), is used to extend the tagging oligonucleotide 402 along theannealed mRNA 408, as shown by the dashed arrow, replicating theexpressed gene portion 414 of the mRNA as a cDNA fragment 416 with thetagging oligonucleotide 402 attached. In many cases, the reversetranscriptase enzyme used will include terminal transferase activity,which will add a series of cytosine residues 418 to the 3′ terminus ofthe tagged cDNA molecule 416. A template switch oligonucleotide 420,having a set of 3′ guanosine residues is then annealed to the terminusof the cDNA molecule, and extended by the reverse transcriptase, inorder to append an additional priming sequence 422 to the end of theresultant tagged cDNA molecule 424. At this point, the taggingoligonucleotide may include the same transformable tagging sequence 410as other tagged mRNA replicate molecules within a given partition, oreven within many or even all of the partitions.

As will be appreciated, with each successive replication of atransformable tagging segment, a new and differently tagged replicatewill be produced. As such, in many cases, it will be desirable tocontrol the number of replication cycles of the transformable sequencetagged mRNAs, e.g., to a single or few cycles of replication, e.g., 1-4cycles, with single cycle replication preferred. Typically, thermalcycling operations may be used to control the number of cycleoperations, e.g., exposing a given tagging operation to only a singlemelting, annealing and extension operation, to ensure that only onetransformed tagged mRNA replicate is produced from each starting mRNAmolecule.

As noted, the tagged cDNA molecule 424 is then subjected to a singlereplication round by priming a DNA polymerization extension from theappended primer region 422, using a DNA polymerase that is unbiased inits incorporation against the transformable nucleotides in the taggingsegment 410 (while maintaining processivity across such nucleotides).Following a single round of replication, the resulting tagged replicatemolecule 428 will include a new, transformed tagging segment 426, thatwill be substantially unique as compared to other replicated taggingsegments present in the same reaction mixture, thus providing a level ofuniqueness to the original molecule, despite being processed in the samemanner as all other molecules in that reaction mixture.

As will be appreciated, the level of uniqueness of a given replicatedtagging segment will depend upon the level of degeneracy, the number oftransformable bases, and the number of molecules within a given process.In some cases, these parameters may be at a level where the moleculeswithin a given analysis are tagged with complete uniqueness, e.g., norepeated transformed tagging elements within a given reaction mixture,while in other cases, the level of uniqueness will be at a level atwhich it may be expected that duplicate copies of a given gene, e.g.,expression products, may be expected to be tagged with a unique taggingelement relative to each other, but that absolute uniqueness may notexist. In other cases, the level of uniqueness will be that a giventagging segment may yield, following a single round of replication, atleast 10 distinct transformed tagging segments (e.g., having at least 10distinct nucleotide sequences), at least 50 distinct transformed taggingsegments, at least 100 distinct transformed tagging segments, at least200 distinct transformed tagging segments, at least 300 distincttransformed tagging segments, at least 400 distinct transformed taggingsegments, at least 500 distinct transformed tagging segments, at least1000 distinct transformed tagging segments, or in some cases, at least1000 distinct transformed tagging segments from a common startingtransformable tagging segment.

Following the single round of replication, the sample may be treated toremove the original tagging oligonucleotides, including the originaltagged cDNA molecule, that contain the transformable tagging segments,in order to prevent the remaining transformable tagging oligonucleotidesfrom participating in subsequent amplification operations and injectingnew, transformed molecules into the analysis. Removal of theseoligonucleotides may be carried out by a number of methods. For example,in some cases, the original tagging oligonucleotides may include a“handle” moiety that facilitates its removal from a reaction mixture,e.g., through affinity purification. Such handles may include, e.g.,specific nucleic acid sequences that may hybridize to solidsupport-bound complementary probe sequences, to remove those from thereaction mixture. Alternatively, the handles may include other affinitybinding reagents, e.g., biotin, avidin, streptavidin, or the like, thatmay be used to pull out the original transformable taggingoligonucleotides from the reaction mixture. Any of a wide variety ofaffinity reagents may be employed in this regard, e.g., nucleic acids,proteins, peptide, antigens, antibodies, or reactive portions of any ofthe foregoing.

In many cases, digestive removal processes may be used, in order toavoid material losses that may accompany the above-describedpurification processes. In particular, processes in which thetransformable tagging oligonucleotides are preferentially digested ordegraded may be used to remove them from participation in subsequentreaction operations. In an example, the original transformable taggingoligonucleotides may include specific regions or bases that allow fortheir selective digestion or removal. By way of example, the taggingoligonucleotides may include uracil-containing bases at one or morepositions within the overall oligonucleotide sequence. Treatment of thereaction mixture with a uracil targeting digestion process, e.g., uracilDNA glycosylase enzyme followed by DNA glycosylase endonuclease VIIItreatment, e.g., USER, then allows targeted digestion of the originaltagging oligonucleotide sequences, while the replicates containing thetransformed tagging segments will contain no uracil containing bases.Alternatively, the tagging oligonucleotides may include specificrestriction endonuclease cleavage sites, which, when contacted with therelevant endonuclease enzyme, results in cleavage of the transformabletagging oligonucleotides. Additionally or alternatively, replicationprocesses following the tagging process may be carried out using primersequences that include 5′ protected groups, such as phosphorothioategroups, such that those replicate molecules produced in a firstreplication round are protected from 5′ to 3′ exonuclease digestion ofdouble stranded DNA substrate, e.g., using a T7 exonuclease, while theoriginating molecules may be subject to digestion. Similarly, taggingoligonucleotides may be provided with other properties rendering themsusceptible to digestion, e.g., incorporating RNA bases, such that thetagging oligonucleotides may be digested using nucleases specific forRNA substrates, e.g., ribonucleases.

In another approach, the tagging oligonucleotides may incorporatesequence components that prevent them from participating in subsequentreplication events after a first round of replication. By way ofexample, the original tagging oligonucleotides may include sequenceelements, such as uracil containing bases that can prevent theirreplication by certain polymerases, e.g., that are present in laterrounds of replication. In a first round of replication following thecreation of the tagged cDNA molecule 424, a heat labile polymerase,e.g., DNA Poll, Klenow, which may be unbiased for the transformablebases in the tagging segment 410, but is capable of processing throughuracil containing bases, is used to carry out a first round ofreplication resulting in creation of the transformed taggedoligonucleotide 428 that includes no uracil bases. Following the firstround of replication, elevation of the reaction temperature to anappropriate melting temperature, e.g., 90 C, will melt the replicatestrand 428 from the original tagged cDNA 424, while also inactivatingthe first polymerase enzyme. A second, heat stable polymerase, alsopresent in the reaction mix, and which is not capable of replicatingthrough uracil containing bases, e.g., archeal polymerases such as 9degrees north, deep vent, and the like, will then remain active insubsequent amplification operations, to selectively amplify thereplicate transformed tagged oligonucleotide 428, while not replicatingany of the original tagging oligonucleotides, e.g., tagged cDNA 424, orany remaining but unincorporated tagging oligonucleotides 402. Inalternative arrangements, these polymerases may be present iteratively.For example, a first uracil processing polymerase is present in thefirst round of replication. Following this first round of replication,this first polymerase may be removed, e.g., by purifying the nucleicacids away from the polymerase, and the second, polymerase, which isincapable of replicating against uracil containing bases may beintroduced. The presence of the uracils in the original tagging moietymay then prevent further replication of the original tagging segment,and consequent generation of new transformed tagging oligonucleotides.Instead, only direct complements/replicates of the transformedoligonucleotides may be created in these subsequent replication rounds.

While the above is described in terms of a single round of replicationof the tagged cDNA molecule 424, it will be appreciated that additionalrounds of replication, e.g., 2, 3, 4, 5, or more, may be practicedwithin the context of the processes described herein, by allowing fordeconvolution of additional tags imparted to a given analysis. Forexample, knowing the number of expected additional unique taggingmolecules added by virtue of additional rounds of transformingreplication, one may account for the additional level of diversity inthe resulting molecules, in order to extrapolate the original number ofstarting molecules.

In some cases, the needed diversity, and thus, the makeup of atransformable sequence segment may be calculated. In particular, tocalculate the effective diversity of a sequence, one may determine it asa function of the level of degeneracy of the transformable bases and thenumber of such transformable bases in each tagging sequence segment.Using the so-called birthday problem one may calculate the expectednumber of molecules that will share the same tag. The effectivediversity can be calculated by first measuring the output of the processwith a detector (e.g., sequencing a population of sequence segmentsincluding the degenerate bases) and counting the frequency of observedbases at each transformable site. One may then use a diversity index tocompute the effective diversity at each site. An example of such a valuemay be the exponent of the Shannon entropy of each transformable base,times the number of such bases in the tag. The ideal, unbiased 4-waydegenerate base has a diversity of 4. A normal, canonical base, incontrast, may have a diversity of 1 (i.e., it will always be observed asbeing itself). Once armed with this (experimentally determined) valuefor a base, and the number of bases, one can map this to the space ofintegers from 1 to N where N is simply the (effective diversity)×(numberof transformable bases). Applying the following formula for counting theexpected number of collisions when sampling from this integer spacegives the expected number of duplicated sequences (note that this isanalogous to the problem of computing the number of collisions producedby a hash function in computer science), where the probability that thekth integer randomly chosen from [1,d] will repeat at least one previouschoice equals q(k−1;d) above. The expected total number of times aselection will repeat a previous selection as n such integers arechosen, equals:

${\sum\limits_{k = 1}^{n}{q\left( {k - {1\text{;}d}} \right)}} = {n - d + {d\left( \frac{d - 1}{d} \right)}^{n}}$

Following creation of the more uniquely tagged replicates of theoriginal sequence segments, the transformed tagged oligonucleotides maybe subjected to additional processing operations in order to facilitatetheir analysis. For example, in some cases, the transformed taggedmolecules may be subjected to amplification, e.g., using PCR, in orderto produce sufficient quantities of molecules for analysis, e.g., usingnucleic acid arrays or nucleic acid sequencing systems.

In the case of PCR amplification, the transformed tagged molecules maybe processed to add amplification priming sequences to one or both endsof the tagged molecules. In some cases, the tagging moiety may includepriming sequences that may be exploited as amplification primers, asdescribed above. Additional priming sequences may be added to theopposing ends of the tagged segment through, e.g., ligation, orpolymerase extension of amplification primers coupled to random primingsequences, providing replicate sequences for amplification.

The use of various approaches for producing amplifiable tagged nucleicacid molecules is described, for example, in published U.S. PatentApplication Publication Nos. 2014/0378345, 2014/0228255, the fulldisclosures of which are hereby incorporated herein by reference intheir entirety for all purposes.

Nucleic amplification is a method for creating multiple copies of smallor long segments of DNA. DNA amplification may be used to attach one ormore desired oligonucleotide sequences to individual beads, such as abarcode sequence or random N-mer sequence. DNA amplification may also beused to prime and extend along a sample of interest, such as genomicDNA, utilizing a random N-mer sequence, in order to produce a fragmentof the sample sequence and couple the barcode associated with the primerto that fragment.

For example, a nucleic acid sequence may be amplified by co-partitioninga template nucleic acid sequence and a bead comprising a plurality ofattached oligonucleotides (e.g., releasably attached oligonucleotides)into a partition (e.g., a droplet of an emulsion, a microcapsule, or anyother suitable type of partition, including a suitable type of partitiondescribed elsewhere herein). The attached oligonucleotides can comprisea primer sequence (e.g., a variable primer sequence such as, forexample, a random N-mer, or a targeted primer sequence such as, forexample, a targeted N-mer) that is complementary to one or more regionsof the template nucleic acid sequence and, in addition, may alsocomprise a common sequence (e.g., such as a barcode sequence). Theprimer sequence can be annealed to the template nucleic acid sequenceand extended (e.g., in a primer extension reaction or any other suitablenucleic acid amplification reaction) to produce one or more first copiesof at least a portion of the template nucleic acid, such that the one ormore first copies comprises the primer sequence and the common sequence.In cases where the oligonucleotides comprising the primer sequence arereleasably attached to the bead, the oligonucleotides may be releasedfrom the bead prior to annealing the primer sequence to the templatenucleic acid sequence. Moreover, in general, the primer sequence may beextended via a polymerase enzyme (e.g., a strand displacing polymeraseenzyme as described elsewhere herein, an exonuclease deficientpolymerase enzyme as described elsewhere herein, or any other type ofsuitable polymerase, including a type of polymerase described elsewhereherein) that is also provided in the partition. Furthermore, theoligonucleotides releasably attached to the bead may be exonucleaseresistant and, thus, may comprise one or more phosphorothioate linkagesas described elsewhere herein. In some cases, the one or morephosphorothioate linkages may comprise a phosphorothioate linkage at aterminal internucleotide linkage in the oligonucleotides.

In some cases, after the generation of the one or more first copies, theprimer sequence can be annealed to one or more of the first copies andthe primer sequence again extended to produce one or more second copies.The one or more second copies can comprise the primer sequence, thecommon sequence, and may also comprise a sequence complementary to atleast a portion of an individual copy of the one or more first copies,and/or a sequence complementary to the variable primer sequence. Theaforementioned operations may be repeated for a desired number of cyclesto produce amplified nucleic acids.

The oligonucleotides described may comprise a sequence segment that isnot copied during an extension reaction (such as an extension reactionthat produces the one or more first or second copies described above).As described elsewhere herein, such a sequence segment may comprise oneor more uracil containing nucleotides and may also result in thegeneration of amplicons that form a hairpin (or partial hairpin)molecule under annealing conditions.

A plurality of different nucleic acids can be amplified by partitioningthe different nucleic acids into separate first partitions (e.g.,droplets in an emulsion) that each comprise a second partition (e.g.,beads, including a type of bead described elsewhere herein). The secondpartition may be releasably associated with a plurality ofoligonucleotides. The second partition may comprise any suitable numberof oligonucleotides (e.g., more than 1,000 oligonucleotides, more than10,000 oligonucleotides, more than 100,000 oligonucleotides, more than1,000,000 oligonucleotides, more than 10,000,000 oligonucleotides, orany other number of oligonucleotides per partition described herein).Moreover, the second partitions may comprise any suitable number ofdifferent barcode sequences (e.g., at least 1,000 different barcodesequences, at least 10,000 different barcode sequences, at least 100,000different barcode sequences, at least 1,000,000 different barcodesequences, at least 10,000,000 different barcode sequence, or any othernumber of different barcode sequences described elsewhere herein).

Furthermore, the plurality of oligonucleotides associated with a givensecond partition may comprise a primer sequence (e.g., a variable primersequence, a targeted primer sequence) and a common sequence (e.g., abarcode sequence). Moreover, the plurality of oligonucleotidesassociated with different second partitions may comprise differentbarcode sequences. Oligonucleotides associated with the plurality ofsecond partitions may be released into the first partitions. Followingrelease, the primer sequences within the first partitions can beannealed to the nucleic acids within the first partitions and the primersequences can then be extended to produce one or more copies of at leasta portion of the nucleic acids with the first partitions. In general,the one or more copies may comprise the barcode sequences released intothe first partitions.

Nucleic acid (e.g., DNA) amplification may be performed on contentswithin fluidic droplets. Fluidic droplets may contain oligonucleotidesattached to beads. Fluidic droplets may further comprise a sample.Fluidic droplets may also comprise reagents suitable for amplificationreactions which may include Kapa HiFi Uracil Plus, modified nucleotides,native nucleotides, uracil containing nucleotides, dTTPs, dUTPs, dCTPs,dGTPs, dATPs, DNA polymerase, Taq polymerase, mutant proof readingpolymerase, 9 degrees North, modified (NEB), exo (-), exo (-) Pfu, DeepVent exo (-), Vent exo (-), and acyclonucleotides (acyNTPS).

Oligonucleotides attached to beads within a fluidic droplet may be usedto amplify a sample nucleic acid such that the oligonucleotides becomeattached to the sample nucleic acid. The sample nucleic acids maycomprise virtually any nucleic acid sought to be analyzed, including,for example, whole genomes, exomes, amplicons, targeted genome segmentse.g., genes or gene families, cellular nucleic acids, circulatingnucleic acids, and the like, and, as noted above, may include DNA(including gDNA, cDNA, mtDNA, etc.) RNA (e.g., mRNA, rRNA, total RNA,etc.). Preparation of such nucleic acids for barcoding may generally beaccomplished by methods that are readily available, e.g., enrichment orpull-down methods, isolation methods, amplification methods etc. Inorder to amplify a desired sample, such as gDNA, the random N-mersequence of an oligonucleotide within the fluidic droplet may be used toprime the desired target sequence and be extended as a complement of thetarget sequence. In some cases, the oligonucleotide may be released fromthe bead in the droplet, as described elsewhere herein, prior topriming. For these priming and extension processes, any suitable methodof DNA amplification may be utilized, including polymerase chainreaction (PCR), digital PCR, reverse-transcription PCR, multiplex PCR,nested PCR, overlap-extension PCR, quantitative PCR, multipledisplacement amplification (MDA), or ligase chain reaction (LCR). Insome cases, amplification within fluidic droplets may be performed untila certain amount of sample nucleic acid comprising barcode may beproduced. In some cases, amplification may be performed for about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20cycles. In some cases, amplification may be performed for more thanabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 cycles, or more. In some cases, amplification may be performed forless than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 cycles.

Following initial processing operations, the resulting library of taggednucleic acid molecules may be subjected to sequencing to determine theoverall sequence of the library molecules. By identifying the number ofdifferent transformed tagging segments, one may infer a quantitation ofthe number of original starting molecules, including determining apredicted or expected number of starting molecules. Quantitation of (orquantifying) starting molecules, as used herein, refers to a generalquantitation, rather than a specific and definitive quantitation. Suchgeneral quantitation may be generally used as a relative metric, e.g.,to compare quantity metrics from two or more samples, the same ordifferent samples but multiple time-points, samples in response tostimuli, etc., or may be used as a general indication of approximatenumbers of starting molecules without requiring a definitive andabsolutely accurate determination of the precise number of molecules.

Kits

Also provide herein are reagent kits and systems useful in practicingthe methods and processes set forth above. As will be appreciated, thekits may generally include various reagents useful in carrying out thesemethods. For example, kits for use in practicing the described processesmay generally include the tagging compositions described above, such as,for example, oligonucleotides comprising the transformable taggingsegments described above. In some cases, the kits may include diverselibraries of such compositions that include large numbers of diverseoligonucleotides that comprise diverse barcode segments in conjunctionwith transformable tagging segments that may be common among some or allof the library members, but that will yield diversity of such taggingsegments when transformed. In some case, these oligonucleotide librariesmay be bund to particles, such as gel beads or microcapsules, and may,in some cases, include additional sequence elements within theoligonucleotides, e.g., sequencer specific priming and/or attachmentsequences, e.g., as described in Published U.S. Patent ApplicationPublication Nos. 2014/0378345, 2014/0228255, 2015/0376700, 2015/0376605,and 2016/0122817, the full disclosures of which are hereby incorporatedherein by reference in their entirety for all purposes.

Oligonucleotides incorporating barcode sequence segments, which functionas a unique identifier, may also include additional sequence segments.Such additional sequence segments may include functional sequences, suchas primer sequences, primer annealing site sequences, immobilizationsequences, or other recognition or binding sequences useful forsubsequent processing, e.g., a sequencing primer or primer binding sitefor use in sequencing of samples to which the barcode containingoligonucleotide is attached. Further, as used herein, the reference tospecific functional sequences as being included within the barcodecontaining sequences also envisioned the inclusion of the complements toany such sequences, such that upon complementary replication will yieldthe specific described sequence.

In addition, the kits may also include other reagents, such as enzymes,used for carrying out the processes described herein, including, forexample, reverse transcriptases, DNA polymerases, e.g., Klenow, DNAPoll, Phi29 and/or archeal polymerases such as 9 degrees north, deepvent, and the like. Other enzymes may likewise be included, such asligation enzymes, USER enzymes, CRISPR-Cas9 related enzymes, PCRamplification enzymes, e.g., Taq polymerases, etc., and the like.

In some cases, the kits described herein may also include reagents andcomponents useful in partitioning sample materials, such as cells,nucleic acids, etc., into individual partitions such as droplets in anemulsion. These reagents and components may include, e.g., partitioningoils, such as fluorinated oils, fluorinated surfactants, andmicrofluidic devices, for use in generating emulsions of partitionedsample materials, reagents and tagging oligonucleotides as describedherein. These components may be provided in conjunction with and/or foruse on appropriate instrumentation systems designed to drive the fluidsthrough the microfluidic devices in order to create the partitionedreagent emulsions as described. Examples of partitioning reagents,microfluidic devices and instrument systems are described in, e.g.,Published U.S. Patent Application Publication Nos. 2010/0105112,2015/0292988, 2014/0378345, 2014/0228255, and the full disclosures ofwhich are hereby incorporated herein by reference in their entirety forall purposes.

The emulsions of the present invention may be formed using any suitableemulsification procedure known to those of ordinary skill in the art. Inthis regard, it will be appreciated that the emulsions can be formedusing microfluidic systems, ultrasound, high pressure homogenization,shaking, stirring, spray processes, membrane techniques, or any otherappropriate method. In one particular embodiment, a micro-capillary or amicrofluidic device is used to form an emulsion. The size and stabilityof the droplets produced by this method may vary depending on, forexample, capillary tip diameter, fluid velocity, viscosity ratio of thecontinuous and discontinuous phases, and interfacial tension of the twophases. Droplets of varying sizes and volumes may be generated withinthe microfluidic system. These sizes and volumes can vary depending onfactors such as fluid viscosities, infusion rates, and nozzlesize/configuration. Droplets may be chosen to have different volumesdepending on the particular application. For example, droplets can havevolumes of less than 1 .mu.l (microliter), less than 0.1 .mu.L(microliter), less than 10 mL, less than 1 mL, less than 0.1 mL, or lessthan 10 pL.

The kits may also include instructions for using the provided reagentsand components in carrying out the processes described herein, as wellas instructions and software for analysis of resulting data. Theinstructions may be printed in one or more documents or providedelectronically, such as in an electronic file or in a user interface(UI), such as a graphical user interface (GUI), on an electronic deviceof a user.

Methods and systems of the present disclosure may be performed by acomputer system that includes one or more computer processors andcomputer memory. Aspects of the systems and methods provided herein canbe embodied in programming. Various aspects of the technology may bethought of as “products” or “articles of manufacture” typically in theform of machine (or processor) executable code and/or associated datathat is carried on or embodied in a type of machine readable medium.Machine-executable code can be stored on an electronic storage unit,such as memory (e.g., read-only memory, random-access memory, flashmemory) or a hard disk. “Storage” type media can include any or all ofthe tangible memory of the computers, processors or the like, orassociated modules thereof, such as various semiconductor memories, tapedrives, disk drives and the like, which may provide non-transitorystorage at any time for the software programming. All or portions of thesoftware may at times be communicated through the Internet or variousother telecommunication networks. Such communications, for example, mayenable loading of the software from one computer or processor intoanother, for example, from a management server or host computer into thecomputer platform of an application server. Thus, another type of mediathat may bear the software elements includes optical, electrical andelectromagnetic waves, such as used across physical interfaces betweenlocal devices, through wired and optical landline networks and overvarious air-links. The physical elements that carry such waves, such aswired or wireless links, optical links or the like, also may beconsidered as media bearing the software. As used herein, unlessrestricted to non-transitory, tangible “storage” media, terms such ascomputer or machine “readable medium” refer to any medium thatparticipates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

Examples

A summary experiment was performed in which a first strand wassynthesized across a sequence containing 1 of 7 transformable ordegenerate bases, where the synthesis was carried out by 1 of 3different polymerase enzymes. The first strand was synthesized using aprimer containing 4 phosphorothioates on the 5′ end of the extensionprimer so that T7 exonuclease may be used to degrade the template strandcontaining the transformable base while leaving the synthesized firststand intact. Sequencing results showed a wide range of incorporationpatterns and polymerase efficiencies across the seven bases and threeenzymes. By embedding the transformable base within a randomer we wereable to identify combinations of flanking bases that maximize theeffective diversity at the transformable base site by, e.g., affectingthe kinetics of the polymerase as it approaches the transformable baseor by affecting stacking interactions in the template-synthesized strandduplex at the transformable base site. In certain cases, while differentconfigurations yielded different levels of diversity, one optimalcombination appeared to include Taq polymerase with one or both of5-nitroindole or deoxyisoguanosine.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method of differentially tagging individualmembers of a plurality of molecular species for quantification,comprising: (a) co-partitioning (i) a plurality of oligonucleotidetagging moieties and (ii) a single cell comprising a plurality ofnucleic acids in a partition; (b) in the partition, releasing thenucleic acids from the single cell; (c) attaching an oligonucleotidetagging moiety of the plurality of oligonucleotide tagging moieties toeach of the plurality of nucleic acids to generate a plurality of taggednucleic acids, wherein the oligonucleotide tagging moiety comprises oneor more degenerate bases having variable complements when processed by apolymerase; and (d) processing the plurality of tagged nucleic acidswith the polymerase to generate a tagged replicate molecule, wherein thetagged replicate molecule comprises random or semi-random complementbases incorporated into a position of the one or more degenerate bases,to distinctly tag each of the nucleic acids.
 2. The method of claim 1,wherein one or more of the one or more degenerate bases comprises 2-waydegeneracy.
 3. The method of claim 1, wherein one or more of the one ormore degenerate bases comprises 3-way degeneracy.
 4. The method of claim1, wherein one or more of the one or more degenerate bases comprises4-way degeneracy.
 5. The method of claim 1, wherein the one or moredegenerate bases are selected from the group consisting of inosine,deoxyinosine, deoxyxanthine, 2′-deoxynebularine, 2′-deoxyguanosine,5-nitroindole, 3-nitroindole, N6-methoxy-2,6-diaminopurine,6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one, and the non-deoxy (orribo) versions of each of the foregoing.
 6. The method of claim 1,wherein the oligonucleotide tagging moiety comprises from 1 to 20transformable nucleotides.
 7. The method of claim 1, wherein theoligonucleotide tagging moiety further comprises one or more additionaloligonucleotide segments.
 8. The method of claim 7, wherein the one ormore additional oligonucleotide segments are selected from the groupconsisting of primer sequence segments, hybridization sequence segmentsconfigured to hybridize to a target sequence, ligation sequence segmentscomprising one or more sequence overhangs and bridging or splitsequences configured to facilitate ligation to a given sequence,sequencer surface attachment segments, and barcode sequence segments. 9.The method of claim 7, wherein the one or more additionaloligonucleotide sequences comprises a primer sequence selected from arandom primer sequence and a sequencing primer.
 10. The method of claim7, wherein the one or more additional oligonucleotide sequencescomprises a hybridization sequence.
 11. The method of claim 10, whereinthe hybridization sequence comprises a poly-T sequence.
 12. The methodof claim 1, wherein the plurality of oligonucleotide tagging moietiescomprise a poly-T sequence segment, and the plurality of nucleic acidscomprise mRNA molecules.
 13. A method of analyzing nucleic acidmolecules, comprising: (a) co-partitioning (i) a plurality ofoligonucleotide tagging moieties and (ii) a single cell comprising aplurality of target oligonucleotides in a partition; (b) in thepartition, releasing the plurality of target oligonucleotides from thesingle cell; (c) attaching an oligonucleotide tagging moiety of theplurality of oligonucleotide tagging moieties to each of the pluralityof target oligonucleotide molecules to generate plurality of taggedtarget oligonucleotides, wherein the oligonucleotide tagging moietycomprises a one or more degenerate bases having variable complementswhen processed by a polymerase; (d) replicating the plurality of taggedtarget oligonucleotides with the polymerase to generate a taggedreplicate oligonucleotide, wherein the tagged replicate oligonucleotidecomprises random or semi-random complement bases incorporated into aposition of the one or more degenerate bases, to distinctly tag each ofthe target oligonucleotides; and (e) sequencing the tagged replicateoligonucleotide, including the oligonucleotide tagging moiety, toidentify the target oligonucleotide molecule.
 14. An oligonucleotidecomposition, comprising an oligonucleotide that comprises a first regionand a second region, wherein the second region comprises a fixedsequence comprising a plurality of variable complement nucleotides,which plurality of variable complement nucleotides is transformable toyield a distinct molecular tag.
 15. A method of quantifying nucleic acidmolecules in a population of identical nucleic acid molecules,comprising: (a) mutating the population of identical nucleic acidmolecules at an expected mutagenesis rate to create a population ofdifferent mutated nucleic acids; (b) sequencing the distinct mutatednucleic acid molecules; and (c) computing a quantification of thenucleic acid molecules in the population of identical nucleic acidmolecules based upon a number of different mutated nucleic acidmolecules.
 16. A method of differentiating amplification products fromtwo or more identical nucleic acid molecules, comprising: (a) subjectingthe two or more nucleic acid molecules to mutagenesis to produce two ormore mutated nucleic acid molecules; (b) amplifying the two or moremutated nucleic acid molecules to generate amplified mutated nucleicacid products; and sequencing the amplified mutated nucleic acidproducts.